This research project addresses the structure-function properties and signaling pathways of the AT1 receptor, which mediates the physiological actions of angiotensin II (Ang II) in the cardiovascular system, adrenal, kidney, brain, liver, and other target tissues. In addition to its specific roles in Ang II target cells, the AT1 receptor serves as a model for the function of other G protein coupled receptors (GPCRs). Our analysis of the roles of specific amino acids in AT1 receptor function has identified residues involved in ligand binding, receptor activation, G protein coupling, and receptor internalization. Recent examples include the importance of two seventh transmembrane domain asparagine, Asn294 and Asn295, in receptor activation and ligand binding, respectively. Also, membrane-proximal amino acids in the carboxyterminal cytoplasmic domain, in particular Phe301, were found to be significant determinants of receptor expression at the cell surface. Some of the functions of GPCRs known to be regulated by agonist-induced phosphorylation of serine/threonine residues in the cytoplasmic tail and/or intracellular loops of the receptor. A highly specific antibody to the AT1 receptor was raised against a fusion protein containing the 92-amino acid C-terminal fragment of the receptor. This antibody permitted the analysis of agonist-induced phosphorylation in the native AT1 receptor immunoprecipitated from Ang II-treated bovine adrenal glomerulosa cells. Receptor phosphorylation was rapid, and was correlated with the degree of ligand occupancy of the receptor. The AT1 receptor was also phosphorylated by activation of protein kinases A and C, but to a lesser extent than that induced by Ang II. Inhibition of PKC activity by stauresporine abolished TPA-induced receptor phosphorylation but enhanced Ang II-induced phosphorylation. This suggests that PKC may exert an inhibitory action on the G protein receptor kinases that are regarded as the major mediators of agonist-induced receptor phosphorylation. An investigation of the sites at which the AT1 receptor is phosphorylated was performed on mutant receptors bearing deletions or mutations in the cytoplasmic tail. This study utilized epitope-tagged AT1 receptors that were expressed in COS cells and immunoprecipitated with an anti-HA antibody after agonist stimulation. The Ang II-induced phosphorylation of the AT1 receptor was found to be confined to an 11-amino acid serine/threonine-rich region in the C-terminal cytoplasmic tail, indicating the importance of this region in receptor internalization and